Hybridoma cell strain capable of secreting African swine fever virus p34 protein monoclonal antibody, monoclonal antibody and application

An African swine fever virus, hybridoma cell line technology, applied in the direction of antiviral immunoglobulin, virus/phage, application, etc., can solve the lack of research and development of non-swine fever virus vaccine methods, and no very effective African swine fever has not been developed. virus vaccine and other issues to achieve a strong specific effect

Pending Publication Date: 2020-08-18
嘉铭(固安)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, due to the complexity of AFSV genome and the diversity of African swine fever protein structure, a very effective African swine fever virus vaccine has not been developed, and there is a lack of research on the development of non-swine swine fever virus vaccine methods

Method used

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  • Hybridoma cell strain capable of secreting African swine fever virus p34 protein monoclonal antibody, monoclonal antibody and application
  • Hybridoma cell strain capable of secreting African swine fever virus p34 protein monoclonal antibody, monoclonal antibody and application
  • Hybridoma cell strain capable of secreting African swine fever virus p34 protein monoclonal antibody, monoclonal antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1p34

[0048] Embodiment 1 p34 antigen preparation

[0049] 1. Construction of pSecTag-2B-p34 vector

[0050] 1. Primer synthesis

[0051] p34-HindIII-F cccAAGCTTATGGGGAATCGCGGGTCTTCTA (SEQ ID No. 1)

[0052] p34-Tb-NotI-R atttGCGGCCGCGCTGCCGCGCGGCACCAGGCCCTTTTT

[0053] GGCGCAGCTGTT (SEQ ID No. 2)

[0054] 2. PCR amplification of p34 gene fragment

[0055] PCR reaction system: Q5 Mix 25μL, Primer F&R 2μL, Template 0.5μL (artificially synthesized), replenish water to 50μL; reaction conditions: 98°C 10s (98°C 10s, 60°C 30s, 72°C 1min) × 35 cycles, 72°C 4min , 4°C, ∞; the amplification result is as follows figure 1 As shown, an amplified band was generated at 1100 bp.

[0056] 3. Single enzyme digestion of target fragment and vector

[0057] Enzyme digestion reaction system: 3 μg each of vector and target fragment, 1 μL of NotI; 5 μL of 10×cutsmart buffer; replenish water to 50 μL; among them, the pSecTag 2B vector was purchased from Invitrogen.

[0058] Reaction conditions: 37...

Embodiment 2

[0091] The preparation of embodiment 2 hybridoma cells

[0092] (1) Preparation of immune splenocytes

[0093] 1. Immunization of mice with p34 protein

[0094]The African swine fever virus p34 protein prepared in Example 1 was used as an antigen to immunize BALB / c mice. After the antigen was fully emulsified with the same amount of Freund's complete adjuvant for the first immunization, the mice were subcutaneously injected into the abdomen at a dose of 100 μg / mouse , Negative serum was collected before immunization for use. After 14 days, the second immunization was performed. The same dose of antigen plus an equal amount of Freund’s incomplete adjuvant was mixed and emulsified for the second immunization and injected into mice. After a total of 5 immunizations, blood was collected from the tail vein of the mice, the serum was separated, and the serum antibody titer was detected by ELISA. The antibody titer detection method was as follows:

[0095] 1) Coat p34 antigen with...

Embodiment 3

[0122] The screening of embodiment 3 hybridoma cells

[0123] The hybridoma cells capable of secreting monoclonal antibodies were screened with antigens prepared by three expression systems. The three antigens were p34 antigen expressed by CHO-S cells, p34 antigen expressed by SF9 insect cells, and recombinant adenovirus vector The p34 antigen prepared by pAd5-p34 transfection HEK293 cells.

[0124] Wherein, the process of preparing p34 antigen expressed by SF9 insect cells is as follows:

[0125] The gene sequence expressing African swine fever virus p34 protein was connected to the Bacmid shuttle plasmid pFastBac to obtain the expression vector pFastBac-p34, and the Bacmid competent cell DH10Bac was used to transform pFastBac-p34 into it, and the virus genome was extracted to be Bacmid-p34 , Bacmid-p34 was transfected into SF9 cells, induced to express and purified to obtain African swine fever virus p34 protein. Among them, the Bacmid system was purchased from Invitrogen ...

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Abstract

The invention discloses a hybridoma cell strain capable of secreting an African swine fever virus p34 protein monoclonal antibody, the monoclonal antibody and an application, the preservation number of the hybridoma cell strain is CCTCC NO: C202042, and the preparation method of the hybridoma cell strain comprises the following steps: 1) expressing to obtain an African swine fever virus p34 protein, 2) immunizing animals to obtain immunized spleen cells, 3) carrying out cell fusion and positive hybridoma cell strain screening, and 4) screening a positive hybridoma cell strain secreting a monoclonal antibody, wherein the p34 protein expressed by HEK293 cells transfected with CHO-S cells, SF9 insect cells and recombinant adenovirus vectors is adopted to screen positive hybridoma cell strains. The p34 antigen protein is prepared by adopting a mammalian cell CHO expression system, so that the folding and glycosylation of the protein are closer to those of natural protein, hybridoma cell strains are screened through the antigens prepared by the three expression systems, so that the obtained hybridoma cell strains can stably secrete monoclonal antibodies, and the specificity is stronger.

Description

technical field [0001] The invention belongs to the field of genetic engineering and recombinant vaccines, and in particular relates to a hybridoma cell strain secreting African swine fever virus p34 protein monoclonal antibody, monoclonal antibody and application. Background technique [0002] African Swine Fever (ASF) is a highly contagious porcine viral disease that can cause close to 100% mortality in domestic pigs. ASF is caused by ASF virus (ASF Virus, ASFV). ASFV is a large double-stranded DNA virus that mainly replicates in the cytoplasm of macrophages. 170-190kb, containing 151 open reading frames, encoding 150-200 proteins, double-stranded linear DNA virus with envelope. The structural proteins that make up the virion include P72, P54, pp62, P220 and other proteins, among which pp62 and pp220 are two polyprotein precursors of ASFV, encoded by the CP530R and CP2475L genes, respectively. The pp62 protein is a late protein of viral replication, and after translation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/08C07K14/01C12N15/34C12N15/85C12N15/866C12N15/861G01N33/569G01N33/577C12R1/91
CPCC07K16/081C07K14/005C12N15/85C12N15/86G01N33/56983G01N33/577C12N2710/12022C12N2710/10043C12N2710/14043
Inventor 陈平李娜王暄张婷婷杨文清李娅芳张利娟
Owner 嘉铭(固安)生物科技有限公司
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